This proposal, targeted for the National Human Genome Research Institute, has the goal of funding an R01 program to develop near term technologies to lower the cost of DNA sequencing. As illustrated by the history of DNA synthesis, costs will most likely drop through a stream of "small step" innovations in chemistry, enzymology, and instrumentation. This proposal, from a laboratory with a track record of innovation in nucleic acid chemistry, enzymology and bioinformatics, will provide this stream, in part by combining individual innovations that have emerged from the Benner laboratories, including nucleoside analogs and polymerases that accept them. The innovations are: Artificially expanded genetic information systems (AEGIS), a DNA-like system that forms duplexes following Watson-Crick rules, but has no interaction with natural nucleic acids [Ben04]. A self-avoiding molecular recognition system (SAMRS), a DNA-like molecule whose components do bind natural DNA, but do not bind other components of the same unnatural SAMRS system [Ben07b]. DNA polymerases and reverse transcriptases that accept both AEGIS and SAMRS components. Emulsion-based directed evolution (EBDE) to optimize polymerases that incorporate unnatural nucleotides. Reversible terminators that terminate primer extension, but reversibly, and polymerases that accept them. SNAP2, which allows priming of DNA synthesis with the specificity of a 16mer, but discrimination of 8mers. Naturally organized genomic databases that support primer design and help analyze the biological meaning of sequence data collected. These technologies will be combined to develop (a) SNAP2 primers that incorporate SAMRS components, permitting highly multiplexed priming and PCR with primers having an effective length of 16mers (or longer) without creating PCR artifacts and supporting single nucleotide discrimination, (b) AEGIS tags appended to SAMRS primers that allow binning of the non-repeating elements of a patient's genome on an array without the need for single molecule chemistry, and (c) DNA polymerases that accept as substrates triphosphates that combine a fluorescent tag on their gamma phosphorus units with a 3'-ONH2 reversible terminator. We will benchmark the new combination reagents and their corresponding enzymes to optimize rates of addition of triphosphates in template-directed polymerization reactions, as well maximize discrimination and minimize mismatching. Throughout, we will use the POSaM DNA array synthesizer as a "beta test " platform to examine combination reagents in the context of small-scale "sequencing-during synthesis " tests. The deliverables will be reagents that meet the "wish lists" of NHGRI with respect to cost, helping meet is $100,000 genome sequence goal, with reduced costs likely as further incremental improvements in the chemistry follow. PROJECT HEALTH RELEVANCE The NHGRI needs to have a laboratory developing, in an R01 format, new chemical reagents and enzymes to support sequencing-during-synthesis and other parallel sequencing architectures that will help it achieve is $100,000 cost-per-genome goal. It is unreasonable to expect that a single technological advance will achieve this goal in a single stroke;this is illustrated by experience in the history of DNA synthesis, where costs dropped through innovations in chemistry, enzymology, and instrumentation that continued for decades. Indeed, much of the cost that will be borne by NHGRI centers with sequencing architectures that are existing or under development comes from their application of off-the-shelf reagents rather than innovative sample processing chemistry, much of which is current available, at least in the development stage. This proposal, from a laboratory with a track record of innovation in structures, reagents, and enzymes to manipulate nucleic acids, is focused on near term development of these to enable NHGRI cost-reduction sequencing goals.